Process for manufacturing clavulanic acid salt

ABSTRACT

According to the present invention, a process for manufacturing alkali metal salt of clavulanic acid wherein impure clavulanic acid in aqueous solution is extracted by a solvent mixture of ketone and alkyl acetate under acidic condition, treated in a conventional manner, and a solution of alkali metal salt of alkanoic acid dissolved in ketone or alkanol solvent is added to obtain pure alkali metal salt of clavulanic acid is provided. 
     Since the process according to the present invention omits a step of formation of amine salt, an economic and simple method for manufacturing alkali metal salt of clavulanic acid in a high yield without excessive use of solvent is provided.

TECHNICAL FIELD

The present invention relates to a novel process for manufacturing saltsof clavulanic acid represented by the following formula(I). ##STR1##

BACKGROUND ART

The resistance of β-lactam antibiotics is associated with inactivationof β-lactam structure due to the opening of β-lactam ring by β-lactamaseproduced by bacteria. Though the inactivating enzymes are commonlycalled as β-lactamase, they are largely divided into penicillinase andcephalosporinase, and their characteristics are different from oneanother dependent upon the species of microorganisms.

Clavulanic acid, an inhibitor of the β-lactamases, is produced from theculture solution of Streptomyces clavuligerus. Clavulanic acid itselfhas only weak antibacterial activity, but it potently enhances theactivity of β-lactam antibiotics against many resistant strains ofbacteria. Presently, drugs containing amoxicillin and clavulanic acidhave been widely used and given clinically, since it exhibits potentantibacterial activity against many resistant strains of bacteria.

Clavulanic acid represented by the above formula (I), and salts andesters thereof are disclosed in Belgium Patent No. 827,926. The processof manufacturing clavulanic acid by submerge fermentation ofStreptomyces sp. microorganisms such as Streptomyces clavuligerus,Streptomyces jumonjinensis and Streptomyces katsurahamanus has beendescribed in British Patent No. 1,508,977.

A variety of methods to recover clavulanic acid from the fermentationsolution are stated in the above-mentioned British Patent. According tothose methods, some standard techniques such as solvent extraction ofaqueous clavulanic acid and ion exchange chromatography were employed.Nevertheless, those methods are disadvantageous in that the low-purityfinal product containing a small amount of toxic impurities has obtainedoccasionally, thus a novel method for manufacturing pharmaceuticallyacceptable clavulanic acid and salts thereof with non-toxicity and highpurity is still required.

Meantime, another method of manufacturing potassium clavulanate fromtertiary butylamine salt, an intermediate of clavulanic acid, wasdescribed in European Patent No. 0,026,044. According to the method,crude clavulanic acid was extracted with ethyl acetate, and the samevolume of acetone was added as a co-solvent and then tertiary butylamineadded thereto so as to crystalize the tertiary butylamine salt ofclavulanic acid to isolate the salt as an intermediate. However, largeamount of solvent is required in the process because large amount ofacetone should be added to the ethyl acetate extract prior to thetreatment with tertiary butylamine, and acetone solvates of variouscomposition between 2% and 8% in proportion to the tertiary butylamineare formed. In addition, severe disadvantage is involved due to the useof tertiary butylamine which is highly toxic (LD₅₀ to rats in case oforal administration: 180 mg/kg) and volatile (b.p: 44.4° C., 760 mmHg)"Dangerous Properties of Industrial materials", fifth ed., N. IrvingSax, and "Chemical Rubber Company Handbook of Chemistry and Physics",53rd. ed., 1972-1973, page 400!. Therefore, the use of tertiarybutylamine in an industrial scale may cause a danger to the workers ofthe industry.

Furthermore, since tertiary butylamine is water-soluble in allproportions, it is difficult to be recovered from the aqueous wasteafter the removal of the solvents. This may cause an economicaldisadvantages as well as possible contamination problems owing to thetertiary butylamine contained in the waste fluid of the factory.Further, in case that a large amount of solvent mixture is used asdisclosed in the said patent, an additional process for recoveringsolvent is required so that enormous economic loss occurs.

Under these circumstances, use of other amines and alternative solventsystems have intensively been studied. For example, several amine saltsof clavulanic acid have been described in the patent literature ofGB-A-1508977, BE-A-862211 and GB-A-1543563. According to the saidliteratures, it is noted that amine salt of clavulanic acid is formedwith secondary or tertiary amines, or primary amines of which the sidechain contains secondary or tertiary alkyl unit. However, most of theseamines are inadequate for manufacturing clavulanate salts or to be usedas an intermediates for the manufacture thereof due to the formation ofamine salts of clavulanic acid having hygroscopicity and/or toxicity.

Further, another process for manufacturing clavulanate salt fromtertiary octyl amine salt having relatively low toxicity and volatilitywas described in British Patent No. 2,264,944. Nevertheless, the processalso has some defects in that the process still is complicated because aprocess for the formation of amine salt of clavulanic acid as anintermediate step for obtaining a pharmaceutically acceptable clavulanicacid; economic or environmental problems may be caused due to the use oflarge amount of solvent; and recovery of the solvent is not easy.

DISCLOSURE OF INVENTION

The present inventors have, through intensive studies, succeeded indeveloping a simple method, free from the said defects of the priorarts, for manufacturing clavulanic acid and its pharmaceuticallyacceptable salts without forming amine salt of clavulanic acid.

Thus, the present invention pertains to a process for manufacturingalkali metal salts of clavulanic acid wherein clavulanate anion from theculture of mutant strain called Streptomyces clavuligerus NRRL 3585 isadsorbed on anion exchange resin; the anion is eluted with aqueoussolution of electrolyte to purify the aqueous solution of clavulanicacid; and then the solution extracted with a mixed solvent of ketone andalkyl acetate; and, after conventional treatment, alkali metal salt ofalkanoic acid dissolved in ketone or alkanol solvent is added to theextract to obtain alkali metal salt of clavulanic acid .

During the extraction process, the desirable pH of the aqueous solutionis between 1 and 2 so that clavulanic acid of the aqueous phase could betransferred into the solvent layer, and the extraction is preferablyperformed at a temperature between 0° to 5° C. In order to get apreferable result, the aqueous solution should contain at least 10mg/ml, preferably more than about 20 mg/ml of clavulanic acid therein.

Alkyl acetate solvent used in the extraction process may be selectedfrom the group consisting of ethyl acetate, methyl acetate, propylacetate and n-butyl acetate; among them, the use of ethyl acetate ismore desirable. As a ketone solvent, methyl ethyl ketone or methylisobutyl ketone may be used; but methyl isobutyl ketone is morepreferable.

When a solvent mixture of ethyl acetate and methyl isobutyl ketone isused as a extraction solvent, the content of methyl isobutyl ketone ispreferably 15 to 70%, more preferably 15 to 40%, and most preferably 18to 25%. In case of methyl isobutyl ketone having less than 15% in itscontent, it has been proved that potassium clavulanate can not easily becrystallized. If the content of methyl isobutyl ketone exceeds 70% or itis used alone as a solvent, high boiling point of methyl isobutyl ketonemay lead to a prolonged time of concentration after extraction, and highyield can not be expected.

As an alkanol solvent, n-butyl alcohol is desirably used. As an alkalimetal salt of alkanoic acid, potassium 2-ethylhexanoate is preferablyused.

To adjust pH during the extraction process, inorganic acid, preferablysulfuric acid may be employed.

The afore-mentioned term, "conventional treatment" of the extract meansa process of drying over a drying agent such as anhydrous magnesiumsulfate, filtering or concentrating the extract. It is more desirable toremove colored impurities from the extract by treating with activatedcarbon.

During the process of the present invention, alkali metal salt ofalkanoic acid includes sodium or potassium salt thereof. Among these,potassium 2-ethyl hexanoate is particularly preferred. The moisturecontained in the product thus obtained can be removed by adding acetoneto the alkali metal salt formed henceforth and slurring.

The present invention shortening a step of amine formation requires lessamount of solvent and provide a better yield compared to the existingmethods of manufacturing clavulanic acid salt, as well as it may providean economical and safe method by preventing possible dangers involved inhandling of toxic amine compounds.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention is further described by referring to thefollowing examples. However, the present invention should not beunderstood to be limited to the examples.

EXAMPLE 1

100 ml of the solvent mixture of ethyl acetate and methyl isobutylketone (4:1) already cooled to 5° C. was added to 100 ml of aqueoussolution containing clavulanic acid (12 mg/ml). While agitating themixture, 50% sulfuric acid was slowly added in order to control pH at1.5. After extracting the said mixture, the extracts are separated. Theremaining aqueous solution was further extracted with 100 ml of thesolvent mixture three times. Anhydrous magnesium sulfate (10 g) andactivated carbon(10 g) were added to the combined extract, and theresultant mixture was agitated at 5° C. for 30 minutes. The mixture wassuction-filtered through a filter, and the remaining material was washedwith 50 ml of methyl isobutyl ketone. A solution of potassium2-ethylhexanoate (1.5 equivalent) dissolved in methyl isobutyl ketonewas slowly added to the said extract, and the mixture was agitated at 5°C. for 2 hours. The crystalline product was filtered and slurried withacetone at the same temperature for 2 hours. The crystalline was driedin a vacuum desiccator (30° C.) for about 5 hours to obtain 1.07 g ofpotassium clavulanate (yield: 73%).

EXAMPLE 2

The mixed-solvent extract of Example 1 was concentrated to 10% andtreated with activated carbon, and then agitated at 5° C. for 20minutes. The mixture was suction-filtered through a filter, and theremaining material was washed with methyl isobutyl ketone. A solution ofpotassium 2-ethylhexanoate (1.5 equivalent) dissolved in methyl isobutylketone was slowly added to the said extract, and the mixture wasagitated at 5° C. for 2 hours. The crystalline product was filtered andslurried with acetone at the same temperature for 2 hours and was driedin a vacuum desiccator (30° C.) for about 5 hours to obtain 1.08 g ofpotassium clavulanate (yield:, 74%).

EXAMPLE 3

The reaction was conducted in the same manner as Example 2 except thatn-butyl alcohol was used as a solvent for dissolving potassium2-ethylhexanoate to obtain 1.04 g of potassium clavulanate (yield: 71%).

What is claimed is:
 1. A process for manufacturing alkali metal salt ofclavulanic acid consisting essentially of:extracting impure clavulanicacid in aqueous solution by a solvent mixture of ketone and alkylacetate under acidic condition, wherein the ketone content of thesolvent mixture is 15 to 70%, and adding thereto a solution of alkalimetal salt of alkanoic acid dissolved in ketone or alkanol solvent toobtain pure alkali metal salt of clavulanic acid.
 2. The processaccording to claim 1, wherein the ketone solvent is selected from thegroup consisting of methyl ethyl ketone and methyl isobutyl ketone, thealkyl acetate is ethyl acetate, and the alkanol solvent is n-butylalcohol.
 3. The process according to claim 2, wherein the ketone contentof the solvent mixture of ketone and alkyl acetate is 15 to 40%.
 4. Theprocess according to claim 1, wherein the alkali metal salt is potassiumsalt.
 5. The process according to claim 1, wherein the alkali metal saltof alkanoic acid is potassium 2-ethylhexanoate.
 6. The process accordingto claim 2, wherein the alkali metal salt is potassium salt.
 7. Theprocess according to claim 2, wherein the alkali metal salt of alkanoicacid is potassium 2-ethylhexanoate.